Routine Drug Testing Research Articles

Zuclopenthixol decanoate toxicity in an 8‐month‐old colt causing extrapyramidal neurological signs

SummaryZuclopenthixol decanoate is a dopamine antagonist licensed for human use to manage schizophrenia and paranoid psychosis. It is in the same drug class (thioxanthene) as fluphenazine decanoate, a drug whose use in horses is reported anecdotally for providing long‐acting sedation. This case study describes severe extrapyramidal signs seen in a Thoroughbred colt after zuclopenthixol decanoate administration. The colt was presented to a referral hospital for an unusual manifestation of colic signs. On admission, the colt began to show clear extrapyramidal neurological signs. The initial suspicion was of fluphenazine decanoate toxicity, which in the limited published literature is treated with intravenous formulations of diphenhydramine hydrochloride and benztropine mesylate. At the time of writing both were unavailable in the United Kingdom without an import license. The closest alternative option, oral diphenhydramine was administered alongside intravenous atropine and pergolide. Improvements in the colt's demeanour and neurological status was noted within 24 h. The colt was hospitalised for a total of 26 days and at time of discharge was clinically normal. Follow‐up communication 3 months after discharge confirmed that the colt had remained neurologically normal. Crucially, toxicology testing submitted to Cornell University was negative for common toxins, including fluphenazine decanoate. Specific secondary testing was performed after suspicion the administration of zuclopenthixol decanoate was raised, which was positive for this compound. Routine drug testing would, therefore, not identify the use of this behaviour modifying drug. Clinical data regarding the use of zuclopenthixol in animals is limited to its use in the translocation of wildlife to relieve stress and aid acclimatisation. To our knowledge, there is no available literature describing the administration of zuclopenthixol decanoate in equine patients.

The surge of bromazolam-related fatalities replacing other novel designer benzodiazepines-related fatalities in San Francisco.

Bromazolam, a novel designer benzodiazepine (NBD), exhibits potent sedative, hypnotic and anxiolytic effects, raising concerns regarding its potential for misuse and fatal outcomes, particularly when combined with opioids such as fentanyl. Despite limited documented fatalities globally, its use poses a significant threat, exacerbated by under-reporting and a lack of routine testing. This study analysed NBD-related deaths in a major US city over a 4-year period. Analysis of accidental overdose deaths involving NBDs in San Francisco, CA, USA from 2020 to 2023, was performed utilizing medico-legal death investigations including comprehensive forensic toxicology, pathology and demographic information. San Francisco conducts thorough investigations into all non-natural and sudden unexpected deaths, including routine alcohol and drug testing of decedents under its jurisdiction, including etizolam, flualprazolam, flubromazolam and bromazolam analysis. There was a sudden surge in bromazolam-related deaths, with 44 fatalities documented in 2023, contrasting with relatively fewer deaths related to other NBDs. Bromazolam fatalities frequently involved co-ingestion with opioids, primarily fentanyl, and stimulants such as methamphetamine and cocaine. Demographic characteristics indicated a predominance of males, with a significant proportion lacking fixed addresses. Blood concentrations of bromazolam increased during the study period, suggesting heightened availability and/or purity in the community. There was a surge in bromazolam-related deaths during 2023 in San Francisco, CA, USA, contrasting with relatively stable numbers of deaths associated with other NBDs over the preceding years. The findings underscore the urgency for enhanced death investigation, testing and reporting to facilitate targeted harm reduction strategies for individuals at risk of bromazolam-related morbidity and mortality.

Appearance of hexahydrocannabinols as recreational drugs and implications for cannabis drug testing – focus on HHC, HHC-P, HHC-O and HHC-H

This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.

Drug War Dragnet

This paper examines the multilayered dynamics behind the drivers of overdose deaths, criminal legal-system involvement, and the drug war infiltration of people’s everyday lives—especially for people under community supervision. While incarceration receives more media and academic attention because of its particular cruelty, almost twice as many people—3.7 million, or one in every sixty-nine U.S. adults—are under community supervision. Probation and parole are commonly understood as “alternatives to incarceration” or “lenient sentences,” but people on supervision must endure constant monitoring, perpetually under the threat of incarceration. Drug war policies and practices have profoundly shaped probation and parole. Regardless of someone’s original sentence, abstinence from drugs, drug testing, submission to warrantless searches, and court-ordered treatment are routine features of supervision. The putative goal of community supervision is to ensure successful reintegration; yet drug war surveillance enacts extensive barriers, while not reducing drug use or drug-related harms like overdose. In order to ensure health, financial security, and overall well-being of those under supervision, policymakers, probation and parole officers, clinicians, service providers, and researchers must work to identify and remove barriers to care, including routine drug testing, substandard or forced substance use disorder treatment, and poor-quality services and support.

‘Spice’ Use Motivations, Experiences, and Repercussions among Veterans of the United States Armed Forces

Background and Objectives The potential for synthetic cannabinoids (SCs) to function as an alternative to marijuana without the same risk of a positive urinalyses led to claims of pervasive military SC use. Case studies confirm use among veterans, but no study has adequately explored SC use in the military using detailed interview data. Methods Interviews (1-2 h) were conducted with 318 justice-involved veterans. Recruitment was attempted with all participants in eight veterans treatment courts in three U.S. states (54.9% of 579 eligible veterans). Interviews were transcribed and thematic analyses completed. Results SC use was reported by 65 participants (21.3%). Major emergent themes indicated SCs were perceived as unpleasant, overly powerful, and a poor substitute for marijuana. Further, habitual use was rare as many chose not to reuse after initial negative experiences. Few indicated that the perception that SCs would not appear on routine military urinalyses enabled their use. Veterans were aware of the changing drug composition and feared “bad batches.” Conclusions SCs were explicitly disliked both independently and relative to marijuana. Nine discussed avoiding positive military drug screens as a consideration, but negative initial experiences generally prevented progression to habitual use. Veterans did not view SCs as a suitable marijuana replacement. Fears that SCs are being used as a marijuana alternative among veterans subject to frequent drug testing appear unfounded. These interviews suggest that routine military drug testing did not motivate individuals to use SCs habitually as a marijuana replacement; however, veterans’ negative interpretation of SC effects contributed to this outcome.

Identification, Isolation, and Characterization of a Novel Degradation Impurity of Busulfan Using Preparative Chromatography, NMR, and LC-MS.

Busulfan is the most effective medication for treating chronic myelogenous or granulocytic leukemia because it has cytotoxic properties that harm or kill hematopoietic cells. It cannot absorb light in the Ultraviolet range due to its structure. Because of this, it is very challenging to quantify using traditional HPLC coupled with UV/Photodiode Array detectors. So, using sodium diethyldithiocarbamate, a derivatization method was developed to quantify related impurities. A significant unknown impurity was identified in derivatized samples of busulfan and a noticeably high percentage level was discovered during routine drug testing. We aimed to isolate, and characterize the unknown impurity observed in the samples and to identify its root cause. Preparative HPLC was used to isolate the unidentified, derivatized impurity, and 1H NMR, 13C NMR, and MS were used to decipher its structural components. The spectral characterization data analysis showed that the unknown impurity was related to busulfan. Additionally, it was noted that the impurity developed as a result of the residual buffer used to prepare the derivatizing reagent. The isolated impurity was found to be same as comparable to that found in busulfan drug substances, according to the results of the characterization tools. An alternative method of reagent preparation was optimized and deemed satisfactory because the buffer used in reagent preparation is the only factor contributing to the formation of impurities. Using cutting-edge analytical characterization tools, it was possible to explain the structural characteristics of an unknown impurity and discover that it was a novel impurity, which undoubtedly contributes to the comprehension of drug substance reaction properties.

Insulin-mimetic peptides in sports drug testing.

Because of its influence on carbohydrate metabolism and, at the same time, anti-catabolic effects, the misuse of the peptide hormone insulin and its synthetic analogs is prohibited in sports at all times according to the regulations of the World Anti-Doping Agency (WADA). The biological effects of insulin and its analogs are mediated through binding to the insulin receptor, which was also found to be activated by different peptides structurally largely unrelated to insulin. Such insulin-mimetic peptides or selective-insulin receptor modulators (SIRMs) represent a novel class of potential performance-enhancing agents, which is currently not explicitly mentioned on the WADA Prohibited List. Within this research project, advanced solid-phase extraction (SPE) and liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a fast, reliable, and specific assay for the detection of the insulin-mimetic peptides S597 and S519 from plasma. Method validation demonstrated a detection limit of 0.5ng/mL and successfully illustrated the applicability of the approach to routine sports drug testing programs. Moreover, sophisticated and comprehensive in vitro metabolism experiments were conducted, and several metabolic degradation products were identified, which will enhance the information generated from future analyses of doping control samples.

A Systematic Review of Pattern and Prevalence of Cannabis Use among Transporters and Associated Road Traffic Accidents

Background: Cannabis (marijuana), while being prohibited in many countries, is the most commonly used illicit drug worldwide (WHO, 1997). "Cannabis-impaired driving" refers to the impairment brought on by 9-tetrahydrocannabinol's (THC) cognitive and psychomotor effects, which have a negative impact on a driver of a motor vehicle after THC ingestion. In contrast, a "cannabis-positive driver" is a person who operates a motor vehicle while exhibiting driving impairments due to any measurable THC concentration in blood, oral fluid, or urine. A driver is considered to be "driving under the influence of cannabis" (DUIC) if their cognitive or psychomotor abilities are significantly impaired and their blood, oral fluid, or urine contains a specific amount of THC.&#x0D; Aim: This study aimed at conducting a systematic review of pattern and prevalence of cannabis use among transporters and the associated road traffic accidents.&#x0D; Design: A systematic review.&#x0D; Data Sources: Systematic search for worldwide published literature from PubMed, PsycINFO, EMBASE, Google Scholar, Science Direct, Cochrane Library and Medline databases.&#x0D; Study eligibility criteria: These studies provided techniques and/or measurements of the frequency and pattern of cannabis use among transporters and the related traffic incidents.&#x0D; Data extraction: The first reviewer extracted the data, and the second reviewer verified it. Both reviewers individually critiqued each of the identified papers. Data was extracted from each eligible study (including author, title, year, and study setting) following the Preferred Reporting Items on Systematic Reviews and Meta-Analysis (PRISMA). These data abstraction forms were examined, and studies that satisfied the criteria were added to the meta-analysis.&#x0D; Findings: Only 10 studies met all of the inclusion criteria out of the 2,251 papers that were recovered after looking at the titles and abstracts (where an abstract was not available, the article was still counted).&#x0D; Conclusion: The study emphasizes the significance of education, law enforcement, and routine drug testing, reducing bribery among federal prosecutors, and influencing the accessibility of cannabis and other psychiatric substances as assessments for preventing substance cruising and restricting road traffic accidents. It also highlights areas where the study's findings and the scientific literature on risk factors for traffic accidents and preventative measures overlap. To increase acceptance and improve results, it is crucial to incorporate lay perspectives into road safety policies and initiatives.

Myostatin inhibitory peptides in sports drug testing.

Across species, skeletal muscle mass is negatively regulated by the TGF-β cytokine myostatin (MSTN). Inhibitors of this growth factor and its signaling pathways are therefore not only promising therapeutics for muscular diseases but also potential performance-enhancing agents in sports. Within this study, protein precipitation and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a detection method for six novel MSTN inhibitory peptides derived from the regulatory MSTN propeptide and the natural MSTN inhibitor follistatin (FST) from doping control serum samples. The approach was comprehensively characterized and found to allow for a specific detection down to concentrations of 3-9ng/mL. Moreover, several potential metabolites of the drug candidates referred to as DF-3, DF-25, and Peptide 7 were identified as valuable complementary analytical targets for doping control analytical assays. Overall, the acquired data pave the way for an implementation of MSTN inhibitory peptides into routine sports drug testing. Even though no drug candidate has obtained clinical approval yet, a proactive development of detection assays is of utmost importance to deter athletes from misusing such compounds, which are readily available for research purposes and on the black market.

Do dried blood spots have the potential to support result management processes in routine sports drug testing?-Part 3: LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation.

Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete's exposure to doping agents. However, several organizational aspects have to be considered before implementation, such as the verification of the sample collections time point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC-HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a 3-month period (sample collection time points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature [RT], 4°C and -20°C). Prior to experimental analysis, LC-HRMS/MS method characteristics were successfully assessed, including intraday precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after being stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti-doping.

Urine Drug Tests Indicate Higher Prevalence of Combined Alcohol and Cocaine Use Compared to Alcohol Together with Cannabis or Amphetamine-A Possible Link to Cocaethylene.

This retrospective study examined the prevalence of combined ethanol and cocaine use, which produces an enhanced psychoactive effect through formation of the active metabolite cocaethylene, compared to combined use of ethanol and two other common recreational drugs, cannabis and amphetamine, based on urine drug test results. The study was based on >30,000 consecutive samples from routine urine drug testing in 2020, and 2627 samples from acute poisonings in the STRIDA project (2010-2016), in Sweden. Drug testing for ethanol (i.e. ethyl glucuronide and ethyl sulfate), cocaine (benzoylecgonine), cannabis (Δ9-THC-COOH) and amphetamine was done by routine immunoassay screening and LC-MS/MS confirmatory methods. Seven samples testing positive for cocaine and ethyl glucuronide were also analyzed for cocaethylene by LC-HRMS/MS. Among routine samples for which testing of ethanol and cocaine had been requested, 43% tested positive for both substances, compared with 24% for ethanol and cannabis and 19% for ethanol and amphetamine (P < 0.0001). Among the drug-related intoxications, 60% of cocaine-positive samples were also positive for ethanol, compared to 40% for cannabis and ethanol and 37% for amphetamine and ethanol. Cocaethylene was detected (range 1.3-150μg/L) in all randomly selected samples testing positive for ethanol and cocaine use. These results, which were based on objective laboratory measures, indicated that combined ethanol and cocaine exposure was more prevalent than expected from drug use statistics. This may relate both to the common use of these substances in party and nightlife settings, and the amplified and prolonged pharmacological effect by the active metabolite cocaethylene.

Pharmacology, prevalence in Germany, and analytical data of cyclobutylmethyl- and norbornylmethyl-type synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC-like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to the emergence of exploratory SCRAs in 2019-2021. The NBM SCRAs were detected post-amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared with Δ9 -tetrahydrocannabinol and CP-55,940. The CBM SCRAs showed binding affinities of Ki : 29.4-0.65 nm and potencies of EC50 : 483-40.1nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (Ki : 1.87-0.25 nm), with indazole being the most affine. Functional activity data confirmed that the indazole derivative tends to be the most potent of all three NBM SCRAs (EC50 : 169-1.78 nm). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The 'life cycle' of all SCRAs on the drug market was less than a year. Notably, Cumyl-CBMICA was the most prevalent while also having the weakest cannabimimetic properties. Quantification of Cumyl-CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborates the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to remaining stocks.

Acute Kidney Injury (AKI) in Young Synthetic Cannabinoids Abusers.

Background. Synthetic cannabinoid-related acute kidney injury represents an increasingly important public health issue due to the diagnostic challenges given by low clinical suspicion of the disease and the frequent undetectability in routine drug tests. Methods. A systematic literature search on PubMed was carried out until 31 January 2022. Case reports, case series, retrospective and prospective studies, as well as reviews on acute kidney injury related to the consumption of synthetic cannabinoid were searched. Results. The systematic review process selected 21 studies for a total of 55 subjects with synthetic cannabinoid-induced acute kidney injury. Renal damage was demonstrated by elevated serum creatinine levels in 49 patients (89%). On renal ultrasound, the most frequent finding was an increase in cortical echogenicity. Renal biopsy, performed in 33% of cases, revealed acute tubular damage, acute tubulointerstitial nephritis, and acute interstitial nephritis, in decreasing order of frequency. Conclusion. Prompt identification and treatment of synthetic cannabinoid-related acute kidney injury represent a sensitive public health goal both for the acute management of damage from synthetic cannabinoids and for the prevention of chronic kidney disease.

Abstract 1610: Primary cultures from malignant pleural effusions and ascites for drug screening in personalized therapy

Abstract Background: A significant percentage of patients (p.) with solid tumors present with pleural effusion or ascitic fluid during disease history. Although isolation of viable malignant cells from these fluids for drug screening and other experimental purposes have been described in the literature, the success rate in obtaining pure cultures is low and the technique is rarely employed in the clinical setting. Here, we present the results of the implementation in our hospital of routine primary culture and subsequent drug testing from pleural effusions and ascites. Methods: 22 pleural effusions (PE) and 11 ascitic fluid from 33 p. were collected; three colon, one esophagous, one melanoma, eight ovarian, one pancreatic and 19 lung cancer. Total cells were isolated by centrifugation, erythrocytes discarded by density gradient and the remaining cells cultured in RPMI + 20%FBS. Primary cultures were genotyped by next generation sequencing (NGS), FISH, qPCR and nCounter. The antitumor effects of several drugs were tested by MTT, including tyrosine kinase, PARP and KRAS inhibitors and chemotherapeutic agents such as cis-platinum or pemetrexed. Results: Primary cultures were attempted from 33 malignant pleural effusions and ascites samples. Cells grew ≥3 passages and were genotyped in 28/33 cases (84%). Eleven primary cultures were pan-negative by NGS, suggesting that they derived either from non-tumor cells or from a minor sub-clone within the tumor. In contrast, 17 primary cultures showed alterations in oncogenes or tumor supressor genes, allelic fractions were ≥70% in 11 cases. Synchronous liquid biopsies or FFPE biopsies were available for the 17 primary cultures; the same genetic alterations were present in all cases. Five primary cultures with drivers at ≥70% allelic fraction (KRAS G12C; ALK and ROS1 fusions; MET and FGFR1 amplifications) were used for MTT assays. In the three cases where the patient was administered the same drugs tested in primary cultures, results were concordant. Conclusions: Primary culture of pleural effusions and ascites can be implemented in the clinical setting with a significant success rate. Drug testing in primary cultures can be of help in treatment selection. Citation Format: Cristina A. Aguado, Beatriz Garcia, Andrés Aguilar-Hernández, Alejandro Martínez-Bueno, Marta Vives-Usano, Florencia Garcia-Casabal, Ruth Román, Ekaterina Meshoulam, Erika Aldeguer, Nuria Jordana-Ariza, Juan José García-Mosquera, Carlos Cabrera, Santiago Viteri, Sonia Rodríguez, Laura Berrocal-Gómez, Pablo Rubinstein, Clara Mayo-de-las-Casas, Rafael Rosell, Miguel Ángel Molina-Vila. Primary cultures from malignant pleural effusions and ascites for drug screening in personalized therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1610.

Evidence of Designer Benzodiazepine Use in Routine Healthcare Urine Drug Specimens.

The illicit drug market continuously evolves, with new substances introduced to mimic prescription or other illicit drugs while evading detection by routine drug testing. The objective was to determine if designer benzodiazepines would be present in urine samples collected from patients in various healthcare settings. Samples for which providers ordered testing for prescription benzodiazepines during the study period were diluted, subjected to enzymatic hydrolysis, and analyzed using liquid chromatography- tandem mass spectrometry. In addition to prescription benzodiazepines, samples were also analyzed for presence of any of the following designer benzodiazepines: etizolam, diclazepam, delorazepam, lormetazepam, flubromazepam, flubromazolam, and phenazepam. Of 38,073 samples tested, 40 samples contained a designer benzodiazepine and/or a metabolite. Of the 40 samples, 19 (47.5%) also tested positive for a prescription benzodiazepine. Twenty-one samples (52.5%) did not test positive for a prescription benzodiazepine, which would result in undetected benzodiazepine use had only traditional definitive testing methods been employed. Thirty-three (82.5%) samples contained an opioid, including 22 (55%) positive for buprenorphine and/or methadone. The potential harms from the use of designer benzodiazepines are widely unknown due to the lack of traditional pharmacokinetic studies and good manufacturing processes. Our analysis shows that when a designer benzodiazepine was present, over 80% of samples also contained an opioid or a prescription benzodiazepine, which may increase the risk of a drug interaction or adverse drug event. Providers may benefit from knowledge of their patients' designer benzodiazepine use when formulating risk mitigation strategies as part of a treatment plan.

A Quantitative LC-MS/MS Method for the Detection of 16 Synthetic Cathinones and 10 Metabolites and Its Application to Suspicious Clinical and Forensic Urine Samples.

Background: Synthetic cathinones currently represent one of the most predominant (sub)-classes of new psychoactive substance (NPS) in illicit drug markets. Despite the increased concerns caused by the constant introduction of new analogues, these drugs are not commonly assayed in routine drug testing procedures and may not be detected in standard screening procedures. This study presents a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the detection and quantification of 16 synthetic cathinones and 10 metabolites in human urine. Methods: The method was validated for all analytes using published guidelines. The evaluated parameters achieved acceptable values according to the set criteria. Potential abuse of synthetic cathinones was investigated in suspicious urine samples from Saudi Arabia originating from workplace drug testing, pre-employment and Accident & Emergency (A&E). Such samples generated a presumptive positive immunoassay for amphetamine; however, they yielded a negative LC–MS/MS confirmation for this analyte, following the recommended cutoff values of Substance of Abuse and Mental Health Services Administration (SAMHSA) guidelines. Results: 5.8% of the analyzed samples were found to contain at least one target analyte, namely mephedrone and N-ethylpentylone, as well as their dihydro-metabolites. The results also revealed polydrug use with the synthetic cathinones being present together with other classical stimulant drugs. Conclusions: This is the first report of NPS use in Saudi Arabia with respect to designer stimulant drugs. Confirmatory urine analyses for suspicious stimulant use should extend beyond classical stimulants to cover a broad range of NPSs and their metabolites in order to report any otherwise potentially undetected/new analyte.

Toward Routine Analysis of Anabolic Androgenic Steroids in Urine Using Ion Mobility-Mass Spectrometry.

Anabolic androgenic steroids (AAS) make up one of the most prevalent classes of performance-enhancing drugs banned by the World Anti-Doping Agency (WADA) due to the competitive advantage they can afford athletes. Mass spectrometry-based methods coupled with chromatographic separations have become the gold standard for AAS analysis because of the superior sensitivity and selectivity provided. However, emerging analytical techniques including ion mobility spectrometry (IMS) have been demonstrated in recent applications as a means to further characterize and identify potential unknowns while simultaneously delivering improved sensitivity by filtering noise. Herein we outline the next crucial steps in bringing IMS to the routine drug testing workflow by combining it with established chromatographic and mass spectrometry methods (i.e., LC-IM-MS) for the detection of AAS in human urine. In addition to robust measurement of collision cross sections which can be used for identification purposes, functional group microtrends provide a structural basis on which to elucidate the structure of future novel anabolic agents. Lastly, the developed workflow is tested by analysis of testosterone in a realistic matrix (human urine) and demonstrates a limit of detection of 524 pg/mL, which surpasses the WADA Minimum Required Performance Levels for anabolic steroids. This work is expected to pave the way toward routine incorporation of IMS into analytical drug testing workflows to augment both qualitative and quantitative measure of performance enhancing drugs in the future.

The metabolism of the synthetic cannabinoids ADB-BUTINACA and ADB-4en-PINACA and their detection in forensic toxicology casework and infused papers seized in prisons.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50 , 11.5nM and ADB-4en-PINACA (EC50 , 11.6nM) are similar to that of MDMB-4en-PINACA (EC50 , 4.3nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.

Analytical and medico-legal problems linked to the presence of delta-8-tetrahydrocannabinol (delta-8-THC): Results from urine drug testing in Sweden.

During routine urine drug testing for cannabis use targeting delta-9-tetrahydrocannabinol carboxylic acid (delta-9-THC-COOH) at the Karolinska University Laboratory in Sweden, an unknown interfering peak was observed in the liquid-chromatographic-tandem mass-spectrometric (LC-MS/MS) confirmative analysis. The peak showed the same exact mass and most abundant fragments as delta-9-THC-COOH but a slightly shorter retention time, thereby not fulfilling all requirements for a positive identification. The analytical results suggested that it was a similar compound, and with access to reference material, it could be identified as the double bond isomer delta-8-THC-COOH. Delta-8-THC has recently become popular as a recreational drug, although its legality varies and is sometimes unclear. In Sweden, all THC isomers are classified substances. The slight difference in retention times was sufficient to distinguish the THC-COOH isomers in the routine LC-MS/MS method, but another LC method allowed better peak separation and individual quantification. At the Karolinska University Laboratory, delta-8-THC-COOH was first observed in April 2020, and the highest incidence was noted in June 2020 when it was present in 5.3% of all THC-COOH-positive samples. The incidence later decreased to today only occasional findings. Large differences in the relative presence of the isomers in the urine samples indicated different origin, for example, synthetically produced pure delta-8-THC, or mixtures of both THC isomers formed during combustion of cannabidiol (CBD). In conclusion, the appearance of delta-8-THC and other isomers on the recreational drug market risks causing analytical and medico-legal problems, due to confusion with delta-9-THC.

P68.05 Human Lung Carcinoma Primary Cultures From Malignant Pleural Effusions as a Tool for Drug Screening and Personalized Therapy

In recent years, new therapies have increased the number of treatment options available to the oncologist. However, the type and duration of response is still unpredictable in a significant number of patients and drug testing of patient-derived tumor cells can have clinical utility in this setting. A significant percentage of lung cancer (LC) patients show pleural effusion and viable malignant cells can be found in this fluid. Here, we present the results of the implementation of routine primary culture and drug testing from pleural effusions.